IFG−AG and RSFCIFG−IPL  in the high-creative group had positive relationship with their AUT performances , but not found in the low-creative group. Furthermore, the RSFC changes of DMN did not present significant relationships with AUT performances. The findings not only confirmed the possibility of brain dynamic reconfiguration following divergent thinking, but also suggested the distinct IFGN reconfiguration between individuals with different creativity levels.Evidence suggests divergent thinking is the cognitive basis of creative thoughts. Neuroimaging literature using resting-state functional connectivity (RSFC) has revealed network reorganizations during divergent thinking. Recent studies have revealed the changes of network organizations when performing creativity tasks, but such brain reconfigurations may be prolonged after task and be modulated by the trait of creativity. To investigate the dynamic reconfiguration, 40 young participants were recruited to perform consecutive Alternative Uses Tasks (AUTs) for divergent thinking and two resting-state scans (before and after AUT) were used for mapping the brain reorganizations after AUT. We split participants into high- and low-creative groups based on creative achievement questionnaire (CAQ) and targeted on reconfigurations of the two brain networks: (1) default-mode network (DMN) and (2) the network seeded at the left inferior frontal gyrus (IFG) because the between-group difference of AUT-induced brain activation located at the left IFG. The changes of post-AUT RSFCs (DMN and IFGN) indicated the prolonged effect of divergent thinking. More specifically, the alterations of RSFC Creativity is the foundation of originality, the generation of novel ideas when facing a specific problem  for individual creativity achievements and alternative uses task (AUT) or the Torrance Tests of Creative Thinking (TTCT) for divergent thinking, were developed to probe the underlying mental processes of creativity Plucker, . With thBeyond the perspective of creative-task induction, divergent thinking can be taken as a spontaneous-thought process for idea incubation because of its involvement in the dynamic shifts between memory, emotion and attention  AUT-induced RSFC changes are associated with the creativity performances, and (2) individuals with different levels of creativity use distinct strategy of brain reconfigurations to comply with the challenges of AUT. To test these hypotheses, we recruited forty young participants to perform AUTs and compared their RSFC maps between two rs-fMRI sessions (Pre- and Post-AUT). The levels of creativity were assessed through splitting the participants into two groups (high- and low-creative) based on the CAQ scores and we tested the influence of AUT on RSFC changes for each creativity group.A total of 42 right-handed healthy young adults were recruited in this study. The inclusion criteria were as follows: (a) non-smoking and without drug addiction; (b) not pregnant; (c) no history of neurological and psychiatric disorder history, and (d) no metal body implants. The entire protocol was approved by the Research Ethics Committee of National Taiwan University (NTU-REC No. 201407EM028). Because two participants could not complete the entire experimental procedure, their datasets were excluded in the analysis. Accordingly, the following results are reported for the remaining 40 participants .N = 15, 12 males, mean age = 23.4 ± 3 years, range: 20–30 years). During the rating, the 56 objects were presented consecutively to the participants with inter-trial intervals (ITIs) of 26 s. The participants were instructed to think and express alternative usages for each object within 20 s. The mean number of answer for each object was 2.4. We categorized the difficulty of AUT into three levels as follows: normal, 1.99–2.99 answers per picture; difficult, <1.99 answers per picture; and easy, >2.99 answers per picture. Subsequently, we used the task materials to design the AUT in MRI environments. All of the objects were presented against a white background for both rating and fMRI experiment.The stimuli consisted of 56 grayscale images of objects from daily life with background removed. Prior to the actual fMRI experiment, we rated the number of alternative usages for each object stimulus from a separate group of participants  = 900 ms; repetition time (TR) = 2,000 ms, echo time (TE) = 2.3 ms; flip angle (FA) = 8°; bandwidth = 200 Hz/pixel; NEX = 1. Total scan time was 6 min 14 sec. The fMRI protocol was using a single-shot gradient-echo echo-planar imaging (GE-EPI) sequence with following imaging parameters: TR = 2 s, TE = 34 ms, FA = 84°, bandwidth = 3,005 Hz/pixel, matrix size = 64 × 64 × 33, and FOV = 210 × 210 mm2. Stimuli were presented via E-prime 2.0  with a back-projection projector in 800 × 600 resolution. Participants viewed the stimuli using a mirror mounted on the head-coil and the viewing field was 8.4°  by 6.3°  at a viewing distance of 420 cm. Participants were instructed to respond with a button press using the index and middle fingers of their right hand .MRI experiments were conducted by a 3T MRI scanner  with 20-channel head coil at National Taiwan University. During the experiment, a high-resolution T1-weighted anatomical image was initially scanned using the 3D-MPRAGE sequence with 256 × 256 × 192 matrix size; 0.93 × 0.93 × 0.93 mmDuring the rs-fMRI scans, the participants were instructed to stay still with their eyes open, to not fall into sleep and to not think of anything in particular. The rs-fMRI scan contained 180 measurements (6 min) per session. During the AUTs, the participants were instructed to view gray-scale pictures of various objects from daily life (through a projector), and to think of as many alternative usages as possible within a fixed duration (20 s). Each AUT session contained 12 trials, including one easy-level picture, one difficult-level picture, and ten normal-level pictures to balance the difficulty level, with fixation and cues of 6 s in between; within each trial one single picture was presented for 20 sec. During the 20 s of the picture presentation, the participants were instructed to press the button immediately when they thought of a special usage; and the button-press numbers and the response time were recorded. Each AUT task contained 156 scans (5 min 12 s) in total. After the cessation of each AUT, the participants were asked to recall the answers retrospectively, and the answers were manually recorded by the experimenter. During the 0-back tasks, one English letter was displayed on screen for 0.5 s, followed by a cross fixation for 1.5 s. The participants were instructed to respond using their right hand to press the right button when the letter appearing on the screen was “X”; otherwise, they were to press the left button. The 0-back fMRI contained 152 scans (5 min 4 s) in each session. The datasets generated for this study are available on request to the corresponding author.Beyond the creativity assessment obtained through the CAQ, the two creativity indices of AUT  were also evaluated for each participant according to the scoring method of previous studies  according to their CAQ scores (the cut-off score for the CAQ was 8), and a two-sample t-test was conducted to generate the contrast maps of AUT brain activity.The data were processed using Analysis of Functional Neuro Images (AFNI) Cox, . We procThe rs-fMRI data were processed according to the following steps: (a) field-map correction; (b) motion correction; (c) coregistration; (d) detrending and filtering (0~0.1 Hz); (e) smoothing with FWHM = 5 mm, and (f) normalization; and (g) the physiological noise removal including white matter, cerebrospinal fluid and six motion parameters. Subsequently, the functional connectivity maps before and after AUT were established through the seed-correlation analysis, targeting on two specific brain networks: (1) the bilateral posterior cingulate cortex (PCC)  for assessing DMN  × 2 (pre- and post-AUT) two-way ANOVA (3dMVM), inclusive of the mean framewise displacement to minimize the motion effect on RSFC findings. The multiple comparisons in the group analysis were corrected through the 3dClustSim approach with the auto-correlation function and the significance level was p < 0.05 . To further determine the relationships between creativity scores and the divergent thinking effect of RSFC, the Pearson correlation analysis was used to measure the association between the behavior indices of each individual  and the RSFC changes (Post-Pre) from selected regions of interested (ROIs). The centers of the spherical ROIs (radius = 5 mm) were placed at the peak value of the significant changes in the contrast maps. The effect of divergent thinking was assessed by calculating the RSFC differences between Pre- and Post-AUT after Fisher's z transformation of the correlation coefficients. The z-transformed RSFC changes from the selected ROIs were correlated with the creativity indices  with false-discovery rate (FDR) correction across ROI-wise comparisons. The statistical calculations were performed using SPSS 20 .All group analyses of the fMRI results were conducted on the basis of analysis of variance (ANOVA) and r = 0.30, p = 0.06), without prominent relationships with originality  for all participants. To test the difference in brain activity between high and low creativity individuals, the following results were divided into two groups with equal sample size (n = 20 for each group); where their CAQ scores were 16.6 ± 12.2 for the high-creative group and 5.0 ± 1.9 for the low-creative group. In addition, the two groups showed no significant differences in age (p = 0.06), gender (p = 0.53) and motion during the two rs-fMRI scans (framewise displacement: p = 0.39).The average CAQ score for the 40 participants was 10.8 ± 10.4, ranging from 1 to 62. Regarding the overall AUT performances, the evaluated originality and fluency were 0.33 ± 0.11 and 3.1 ± 1.0, respectively. The CAQ scores were adjusted by the log-transform . t-test (corrected p < 0.05). A difference between the groups was observed only in the left IFG , which was taken as the seed localization for the subsequent RSFC analyses. The AUT-associated brain activations were located at the left superior temporal gyrus, anterior cingulate gyrus, declive of the cerebellum, caudate and occipital visual cortex (p < 0.01). The lower panel of p < 0.01). The IFGN was associated with the bilateral superior frontal, medial frontal, anterior cingulate, insula, superior, and middle temporal gyri. The detailed information of the group × divergent thinking comparisons  for IFGN and DMN is listed in DMN became prominent after AUT engagements, and the RSFCIFGN showed different network reconfigurations between the two groups.To further elucidate the alterations of brain integrity over the divergent thinking process, we applied a seed-correlation approach to investigate RSFC. Two brain networks were targeted with the seed locations in bilateral PCC for DMN and left IFG for IFG-associated network (IFGN) to assess the group (high- vs. low-creative) and divergent thinking (Pre- vs. Post-AUT) effects. The upper panel of IFG−AG: r = 0.51, p < 0.02; fluency-ΔFCIFG−AG: r = 0.49, p < 0.03; originality-ΔFCIFG−IPL: r = 0.63, p < 0.001) in the high-creative group, but the correlations in the low-creative group were non-significant. The Steiger's Z-test presented significant group differences in the correlation coefficients of originality-ΔFCIFG−AG (p < 0.001), fluency-ΔFCIFG−AG (p < 0.004) and originality-ΔFCIFG−IPL (p < 0.001). An ROI analysis was conducted to determine the relationship between RSFC changes and creativity scores. The spherical ROIs were placed according to the results in DMN networks (Pre-AUT) were similar between the two groups, but the baseline RSFCIFGN could reflect the group difference. Then, the IFGN presented significant network reorganization while the DMN did not reorganize after AUT engagements. Intriguingly, such IFGN reconfiguration was associated with AUT performances  could modulate the RSFC of DMN and IFGN, which were associated with creativity performance. The results indicated that the baseline RSFCr = 0.13 for high-creative networks and r = 0.11 for low-creative networks). However, the RSFC–creativity correlation became elevated for high-creative networks when performing the AUT (r = 0.35 and 0.28 for two separate datasets), but the correlation turned out non-significant for low-creative networks during AUT engagements (r = 0.02 to −0.04 for two separate datasets). The evidence implies that dynamic network reorganizations in AUT can be associated with the creativity performances. In this study, assuming that the short-term network reorganizations induced by AUT could sustain after the task cessation (the second resting state in Post-AUT), we demonstrated that dynamic RSFC alterations were associated with the AUT performance . Specifically, we prescribed the ROIs from both the creativity trait effect  in the high-creative group, as well as a significantly positive relationship between originality and the right MTG activity . However, the association between the negative activation and creativity performances is beyond the scope of the current work. Future studies are warranted to discern the underlying mechanism of divergent thinking.As evident in IFGN changes between the two groups in About grouping of CAQ scores, we chose the median split because of a positively-skewed distribution of CAQ in Carson's report. Additionally, the average CAQ score of Carson's report was as high as 14.4, the cut-off line would be around 8 to 9 when we adopted the median split  correlated with the performances of divergent thinking. Furthermore, individuals with different creativity level (high- and low-creative groups) might present diverse alterations of RSFC changes. Before divergent thinking, both high- and low-creative group did not exhibit significant difference of DMN connectivity, but the group difference was highlighted after AUT engagements. Meanwhile, the IFGN indeed presented the network reconfiguration after divergent thinking. Furthermore, the ΔFCsleepbrain@tmu.edu.tw.The datasets generated and analyzed in the current study are not publicly available due to IRB but are available from the corresponding author on reasonable request. Requests to access the datasets should be directed to Changwei W. Wu, The studies involving human participants were reviewed and approved by Research Ethics Committee of National Taiwan University. The patients/participants provided their written informed consent to participate in this study.H-YW: data collection, analysis, and writing. B-CK and C-MH: experiment design and writing. P-JT and A-LH: analysis and writing. L-MH: analysis. C-YL: data collection. J-HC: suggestion of writing. CW: the main idea, experimental design, and writing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Physicochemical characterization of this system includes U-V spectroscopy, dynamic light scattering, Fourier-transform infrared spectroscopy, and transmission electron microscopy. The most stable nanoprobe, in terms of the aggregation, radiolabeling efficiency, and purity, was tested in a sentinel lymph node model in a rat by microSPECT/computed tomography (CT) imaging. The SPECT images revealed that 99mTc-radiolabeled AuNPs functionalized with mannose can track and accumulate in lymph nodes in a similar way to the commercial 99mTc-Sulfur colloid, commonly used in clinical practice for sentinel lymph node detection. These promising results support the idea that 99mTc-AuNPs-mannose could be used as a SPECT contrast agent for lymphatic mapping.Gold nanoparticles (AuNPs) are considered valuable nanomaterials for the design of radiolabeled nanoprobes for single-photon emission computed tomography (SPECT) imaging. Radiolabeled and functionalized AuNPs could improve lymphatic mapping by enhancing the radioactive signaling of individual particles in the sentinel node. In this study, an alternative method for functionalizing commercial AuNps with mannose is described. The chemical derivatization and biofunctionalization of AuNPs were performed with lipoic acid and mannose, respectively. Several levels of mannose were tested; the thiolate hydrazinonicotinamide-glycine-glycine-cysteine (HYNIC) molecule was also used for  Gold nanoparticles (AuNPs) have unique physical and chemical properties that make them valuable nanomaterials for many clinical applications, including their use as contrast media in diagnostic imaging . The bin125I, 111In, 131I, and 99mTc radionuclides have been attached to functionalized AuNPs [125I and 111In) has been used to prepare multimodal probes for SPECT bioimaging [Functionalized AuNPs are ideal contrast agents for hybrid imaging techniques, for instance, single-photon emission computed tomography (SPECT) with computed tomography (CT) . Radiolaed AuNPs ,5,6, whioimaging . In partoimaging ,9. The Soimaging .99mTc is an ideal radionuclide because of its short 6-h half-life and photon energy of 140 keV, as well as its commercial availability and low cost [New contrast agents should improve SLN imaging by enhancing the radioactive signaling of individual particles in the tracking of the lymphatic net and/or increase the radiotracer delivery and retention in the sentinel node . Functiolow cost ,12.99mTc-labeled gold nanoparticles conjugated to HYNIC-GGC/mannose was prepared to evaluate its biological behavior as a potential radiopharmaceutical for sentinel lymph node detection in a rat model [99mTc-AuNP-mannose remains within the first lymph node  of Wistar rats for 24 h, showing the potential of this radioconjugate for the specific targeting of SLN. In the current study, an alternative method for functionalizing commercial AuNPs with mannose (AuNPs-MAN) using lipoic acid is reported. This AuNPs-MAN system was also radiolabeled with 99mTc to evaluate its potential for lymph node mapping using microSPECT/CT imaging in a rat model. Physicochemical characterization of this system includes U-V spectroscopy, dynamic light scattering, Fourier-transform infrared spectroscopy, and transmission electron microscopy. In a previous study, a multifunctional system of at model . It was O-acetyl-α-d-mannopyranoside hydrochloride (MAN) or hydrazinonicotinamide-glycine-glycine-cysteine (HYNIC) did not modify the Z-potential value. Previous studies have shown that AuNPs functionalized with carboxylic acids exhibited negative Z-potential values close to −60 mV [2 (MAN) and –COOH (ALA) moieties would lead to the formation of amide groups. o −60 mV , suggestIncrements in the hydrodynamic diameter were also not statistically significant; in fact, the Polydispersity Index was greater than 0.2 in all groups, indicating the presence of different size populations. It is well-known that the Dynamic Light Scattering (DLS) method gives an estimation of the hydrodynamic diameter of the particle, but it can be greatly affected by the particle shape and structure. In addition, a small portion of large contaminant or aggregated particles can greatly contribute to scattering; it is possible that the inclusion of ALA, MAN, and HYNIC attached to the surface of AuNps affects the estimation of the hydrodynamic diameter, giving information on the AuNp core, along with the coating and the layer of solvent particles attached to its surface . These o48 spectra (red line) show a peak at 1700 cm−1 that was assigned to C=O double bond stretching. The lipid hydrocarbon chains contribute peaks at about 2848 cm−1  and 2920 cm−1 . After the covalent bond (gray line), AuNPs-MAN48 shows a peak at ∼3400 that was assigned to O-H stretching. The peak at 2970 cm−1 was assigned to C-H stretching of the pyranosic sugar rings, while the peak at 1650 cm−1 was assigned to the stretching band of C=O stretching and C-N stretching. Furthermore, the 1580 cm−1 peak was associated with in-plane N-H bending and C-N stretching, while the peak at 1280 cm−1 was related to C-N stretching. Finally, the peak at 1070 cm−1 was related to C-O stretching [max) at about 521 nm. Although the localized surface plasmon resonance (LSPR) spectrum is very sensitive to changes in the nanoparticle’s shape, size, stability, surrounding medium, and/or surface modifications [The UV-Vis spectroscopy analysis of AuNPs-ALA, AuNPs-MAN, and HYNIC-AuNPs-MAN conjugates is presented in ications , the loc48h and AuNPs-MAN72h conjugates were selected for 99mTc radiolabeling. 48h was used for the in vivo SPECT/CT imaging study. According to the DLS, Z-potential and UV-Vis results, the AuNPs-MAN99mTc-AuNPs-MAN48h. After an injection in the footpad, the contrast agent drained from the administration site (AA) into the first lymphatic node, i.e., the popliteal lymph node (PO). Next, the contrast followed the lymphatic drainage toward the iliac lymph node (IL) and then the renal node (RE). During the injection of the contrast agents, it was noticed that the 99mTc-Sulfur colloid induced a significant irritation (observed as a pain reaction in the footpad during and after inoculation); this situation was not observed with the conjugated gold nanoparticles.99mTc-AuNPs-MAN48h was injected in the right footpad, while 99mTc-AuNPs-ALA48h was injected in the left one. Lead covers were placed at the footpads to block the signal from the injection site. The images depict a preferential accumulation of AuNPs-MAN48h in the PO node 3 h post injection. A minimal accumulation of AuNPs-ALA48h was observed in the left PO node, except for the third rat (R3 in the figure). In this rat, accumulation in the IL and right IN nodes was also noticed. These results demonstrate mannose’s ability to promote gold nanoparticle retention within the lymph nodes.48h and AuNPs-ALA48h that stays at the injection site and the popliteal nodes. Values were calculated from the in vivo images and the ex vivo radioactivity quantification after animal sacrifice. Differences between in vivo and ex vivo results were not significant. 99mTc-Sulfur colloid (left footpad) and 99mTc-AuNPs-MAN48h (right footpad). The accumulation of both contrast agents in the popliteal nodes could be observed from the beginning of the study; both contrast agents were retained in the popliteal nodes in different proportions and some moved to the next lymph nodes. Similar behavior was observed in the rest of the rats. This temporal retention of the contrast agent in the lymph nodes means that it can be employed in “radioactive staining” in order to improve SLN detection and identification. A quantitative image analysis is presented in There is current interest in developing new contrast media for sentinel lymph node mapping ,18. In c48 results in hydrodynamic diameters  in the rage of 50–70 nm , Nanocis ; and Hepatate  [99mTc and successfully tested for lymph node mapping by microSPECT/CT in a rat model  with mannose in order to be used in SLN detection. The HRTEM, spectroscopy, and DLS techniques implemented in this study have demonstrated that AuNPs can be successfully functionalized with ALA, MAN, and HYNIC. The TEM results have shown that the average core size of gold nanoparticles does not change throughout the functionalization process ; DLS siz50–70 nm . These s–255 nm) . The mosat model .48 nanoprobes is the standard size of commercial colloids used in SLN detection. To date, there are no commercial contrast agents with an optimal particle range size; however, there is a consensus that the contrast should be small enough to be rapidly removed from the injection site and transported to lymphatic nodes, and yet large enough to be retained for many hours in lymph nodes . Small p99mTc-AuNPs-MAN48 stained the popliteal lymph node  in a similar way to commercial 99mTc-Sulfur colloid, which is commonly used in clinical practice. This result partially supports the potential use of 99mTc-AuNPs-MAN48 in lymphatic mapping. A significant difference observed between both agents was the minimal irritation induced by AuNPs-MAN48 at the injection site compared with the sulfur colloid, which is a common side effect reported in the clinic. Another advantage of mannose-functionalized AuNPs is their potential use for multimodal photoacoustic SLN mapping, where binding to specific macrophage receptors is needed in order improve the specificity of the method [As was shown with the SPECT/CT studies, the conjugate e method ,24. Heree method . This ree method . Becausee method .Finally, a slow rate of movement from the injection site was noticed for both contrast agents . It is i99mTc-AuNPs-MAN) with a high potential for lymphatic mapping using SPECT/CT imaging. In vivo studies have shown that this system can track and accumulate in lymph nodes in a similar way to the commercial 99mTc-Sulfur colloid, which is commonly used in clinical practice for SLN detection. These results support the promising use of 99mTc-AuNPs-MAN in SLN detection; however, more studies are required to evaluate the AuNPs-MAN´s toxicity, biodistribution, and pharmacokinetics before validating its potential clinical use for sentinel lymph node detection and targeting in cancer treatment. In this study, we have reported a simple but efficient method for elaborating a radioactive contrast agent (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), sodium hydroxide (NaOH), ethylenediamine-N,N′-diacetic acid (EDDA), tricine, tin(II) chloride (SnCl2), and hydrochloric acid (HCl) were purchased from Sigma-Aldrich . 2-aminoethyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside hydrochloride (MAN) was purchased from Synthose Inc, . The modified peptide hydrazinonicotinamide-glycine-glycine-cysteine (HYNIC) was acquired from Peptides International . The 99mTc pertechnetate and 99mTc-Sulfur colloid were purchased from Instituto Nacional de Investigaciones Nucleares . All reagents were used as received, without further purification. Deionized water (18.2 MΩ cm) was used in the experiments. Gold nanoparticles (AuNPs) with a 20 nm diameter (stabilized suspension in citrate buffer); lipoic acid (ALA), A450 is the absorption of gold nanoparticles at 450 nm for a standard path length l of 1 cm and Ɛ450 is the molar decadic extinction coefficient at λ = 450 nm for 20 nm gold nanoparticles. These conjugates were labeled according the incubation time, AuNPs-ALA(h), .Pristine AuNPs  in sodium citrate were washed three times  and resuspended in deionized water at pH 11 . Then, 2.5 mL of AuNPs (1 nM) was incubated with 250 µL of ALA (10 mM) for 2, 24, 48, or 72 h at room temperature and constant stirring of 3000 rpm : c = A45CM is the mass concentration of gold (g/mL) and m is the mass of an individual nanoparticle (g/particle).The number of ALA molecules bonded to AuNPs was estimated by a UV-vis calibration curve of ALA concentrations . The specific absorption peak at 330 nm of the five-membered ring in lipoic acid  was sele(h)  was mixed with 2 μL of MAN (100 mg/mL in ethanol) and 10 µL of EDC . These conjugates were labeled as AuNPs-MAN(h) . The solutions were incubated with constant stirring , centrifuged at 11,500 rpm (30 min), washed three times in deionized water at pH 11, and left in basic hydrolysis at pH 11 for 24 h; finally, AuNPs-MAN(h) conjugates were adjusted to a nanoparticle concentration of 1 nM. One milliliter of each AuNPs-ALA(h) (1 nM) was incubated with HYNIC  with constant stirring . The conjugates were labeled as HYNIC-AuNPs-MAN(h) . Unbound HYNIC was removed by ultrafiltration . The nanoparticles were washed three times , resuspended in deionized water at pH 11, and adjusted to a nanoparticle concentration of 1 nM.One milliliter of each AuNPs-MANThe number of HYNIC molecules per AuNP was also estimated by a UV-vis calibration curve from HYNIC . The absorbance peak at 254 nm was used ; the abs(h) were obtained with a Beckman Coulter DU-530 Life science UV/Vis spectrophotometer using a 1 cm quartz cuvette. UV/Vis analysis was used to monitor the AuNP surface plasmon band (520 nm) shift in order to evaluate the conjugation stability. Absorption spectra (400–1000 nm) for each AuNPs-MANThe hydrodynamic diameter and Z-potential were measured by Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering, respectively, using a Z-sizer 90Plus Analyzer . In this equipment, the default calculation employs the Smoluchowski limit to calculate the Z-potential and the Stokes–Einstein equation for particle sizing. Smoluchowski approximation is valid for nanoparticles in aqueous media, even though the smallest nanoparticles (<20 nm diameter) may not have κa >> 1 necessary to justify the application of Smoluchowski approximation . κa >> 1−1, of the covalent reaction for ALA-MAN, were recorded on a Perkin-Elmer Spectrum 100 spectrometer  to corroborate the formation of amide I and II bonds by comparing the spectra of AuNPs-ALA48 before and after its conjugation with MAN. Samples were deposited on the ATR crystal by the drop casting technique and deposition was repeated several times.The attenuated total reflection Fourier transform infrared (FTIR-ATR) spectra, from 1000 to 3800 cmMorphology and chemical composition analysis were performed using a JEM-2010F FASTEM instrument  coupled to a NORAN energy dispersive spectrophotometer (EDS) operating at 20 kV. Samples were prepared by applying 30 μL of a diluted nanoparticle solution onto carbon-coated copper grids . Excess solution was removed with filter paper and the sample was allowed to dry at room temperature overnight. The resulting images were analyzed using ImageJ version 1.40 software ; for size analysis, at least 100 nanoparticles were evaluated per sample and the statistical analysis was performed with OriginPro2020 software .99mTc pertechnetate (1 mCi) with 60 μL of SnCl2  and 2 µL of HCl (12 M) were incubated at 30 °C under constant stirring . Then, the pH was adjusted to 7  for further adding 40 µL of tricine  at 30 °C under constant stirring . Finally, 1 mL of each HYNIC-AuNPs-MAN(h) (1 nM) and 10 μL of EDDA  were added to the previous solution and incubated for 20 min at 100 °C under stirring at 500 rpm. Purification was performed by filtration using Amicon centrifugal filters  at 12,000 rpm, for 5 min. Radiolabeled nanoparticles (99mTc-AuNPs-MAN(h)) remained in the filter, while free pertechnetate (99mTcO4−) and hydrolyzed/reduced technetium (99mTcO2) passed through the filter. The radiolabeling efficiency was evaluated by measuring the radioactivity (mCi) in the supernatant and pellet, using a dose calibrator .Radiolabeling was performed as previously reported . BrieflyRP) was determined by ITLC-SG . The samples (2 μL) were spotted on ITLC strips and 2-butanone and saline solution (NaCl-0.9%) were used as mobile phases. The 2-butanone was used to evaluate the percentage of free 99mTcO4− and the saline solution was employed to measure the percentage of hydrolyzed/reduced technetium (99mTcO2). The strips were cut in half and the radioactivity in each segment was measured using a well-type gamma counter . The % RP was calculated asB and T represent the radioactivity (cps) measured at the bottom  and top segments , respectively.The radiochemical purity (99mTc-AuNPs-MAN(h) was added to 500 μL of either saline solution or human serum and incubated for 6 h. The stability, as a function of time, was determined by ITLC-SG/NaCl-0.9%, as previously described, after collecting samples (15 μL) at 15 min; 30 min; and 1, 2, 4, and 6h. All measurements were corrected by radioactivity decay.The radiolabeling stability was measured in saline solution (NaCl 0.9%) at room temperature and in fresh human serum (37 °C). A total of 100 μL of Male Wistar rats (250–300 g) were obtained from the UNAM’s Medical School animal facility (Mexico City). Animals were kept in a pathogen-free environment and fed with autoclaved food and water ad libitum. The procedures for care and use of the animals were approved by local institutional Scientific and Ethics Committees at INCan (019/010/IBI) (CEI/1349/18) (2019/0403/CB1) and all applicable institutional and governmental regulations were followed in accordance with the Federal Regulations for Animal Production, Care and Experimentation . The guidelines from the Guide for the Care and Use of Laboratory Animals of the National Institute of Health  were also followed. All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments.99mTc-AuNPs-MAN as a contrast agent for SLN mapping, the foot-draining popliteal lymph node model of rats was chosen. This model is widely used to test diagnostic techniques of metastasis and for drug delivery to lymphatic nodes. In this model, the administration of 99mTc-AuNPs-MAN was performed in the footpad of Wistar rats. It is expected that 99mTc-AuNPs-MAN will drain from the administration area to the first lymphatic node, i.e., the popliteal lymph node. To evaluate the potential of 99mTc-AuNPs-MAN48h vs. 99mTc-AuNPs-ALA48h. This experiment was designed to verify the lymphatic node accumulation of nanoparticles coated with mannose. The other six rats (Group B) were used to evaluate the targeting efficiency of 99mTc-AuNPs-MAN48h vs. 99mTc-Sulfur colloid. This colloid is commonly used in clinical practice for the assessment of lymphatic tracking and sentinel node targeting and was used here as a gold standard.A microPET/SPECT/CT imaging system  was used to analyze the in vivo lymphatic mapping of the radiolabeled conjugates in healthy rats. Nine rats were used in this study, organized into two experimental groups. In the first group (Group A), three rats were used to compare the popliteal node targeting of 99mTc-AuNPs-MAN48, 99mTc-AuNPs-ALA48h, or 99mTc-Sulfur colloid in approximately 35 µL. After the injection, the footpad was gently massaged for one minute to promote the movement of the complex into the lymphatic pathway. For the first experiment (Group A), images were acquired at 1 and 3 h post injection and the animals were sacrificed by cervical dislocation after acquiring the 3 h image; popliteal lymph nodes and the injection site were dissected. The nodes were harvested, weighed, and counted for radioactivity using the well-type gamma counter . For the second experiment (Group B), images were acquired at 1, 3, and 6 h post injection.The radiolabeled complexes were injected subcutaneously into the footpad of rats under anesthesia using a mixture of oxygen/isofluorane at 3%. The footpad of each animal received a single injection (~50 µCi) of All images were reconstructed with Albira’s reconstruction software and the quantification analysis and image processing were performed with PMODE  and Osirix MD (Pixmeo SARL), respectively.p < 0.05. All assays were performed with at least three independent triplicates. The results are expressed as the mean ± SD (standard deviation). Statistical analysis was performed using one-way analysis of variance (ANOVA). Significance was assumed at


In a comparison of the two stress models, IS strongly induced depressive and anxiety features, as indicated by all parameters: behavior test scores , serum corticosterone levels (p < 0.05), immunohistological alterations for serotonin activity (p < 0.05) and microglial activity (p = 0.072). Our results indicate the suitability of IS for the development of animal models of depressive disorders and may reveal the medical impact of social isolation environment in modern society.This study aimed to help to understand the influence of stress on depression, which reflects the social environments of especially solitary life and the increasing prevalence of depressive disorders. To determine the distinguishable features of two-representative animal models of stress-induced depressive disorder, we compared isolation stress (IS) and unpredictable chronic mild stress (UCMS). After 4-week of stress, both models showed significant depressive- and anxiety-like behaviors in an open field test (OFT;  Stressful events are inevitable parts of life in modern society, and they have caused a rapid increase in the number of patients with neuropsychological disorders  vs. psychological stress  in patients with major depressive disorder  are secreted in large amounts by hypothalamic-pituitary-adrenocortical (HPA) axis activation. However, their excessive influx into the brain leads to dysfunction of the neurotransmitter system : the non-stress group, the isolation stress (IS) group, and the UCMS groups.Twenty-four specific pathogen-free C57BL/6N male mice  were purchased from Dae Han Biolink Co., Limited . The mice were housed in plastic cages maintained at 23 ± 1°C with a 12 h light-dark cycle, and they were given free access to food pellets  and tap water. After acclimation for 7 days, the mice were randomly divided into three groups (2 anesthesia. The blood was left for 30 min at room temperature (RT) to clot, and the serum was separated. To analyze histology, the brains of the remaining three mice in each group were removed under transcardial perfusion with heparin (10 U/ml in PBS) and 4% paraformaldehyde (pH 7.4) and were stored in 4% paraformaldehyde solution.The isolation stress procedure was conducted as previously described  and the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health.The open-field test (OFT) was performed as previously described . The behavioral tests were performed by researchers who were blinded to the experimental conditions.Serum corticosterone levels were measured using an Arbor Corticosterone Enzyme Immunoassay Kit  according to the manufacturer’s instructions. The absorbance at 450 nm was measured using a UV spectrophotometer .® 488 donkey anti-goat IgG H&L  secondary antibodies for 2 h at RT. To analyze the Iba-1-positive cells, the sections were incubated with an avidin-biotin-peroxidase complex  for 2 h. The peroxidase activity was visualized using a stable diaminobenzidine solution. To analyze the 5-HT-positive signal, the sections were exposed to DAPI  to stain cell nuclei. Immunoreactivity was observed under an Axio-Phot microscope . The intensity was quantified using ImageJ 1.46 software .For cryoprotection, brains were gradually immersed in 10, 20, and 30% sucrose in 24 h intervals and embedded in OCT compound  with liquid nitrogen. The brains were cut into frozen coronal sections (30 μm) using a Leica CM3050 cryostat and then stored in a free-floating buffer. For immunohistological analysis, the sections were blocked in 5% normal chicken serum (containing 0.3% Triton X-100) for 1 h. After washing, the sections were incubated with primary antibodies against ionized calcium-binding adaptor molecule 1  or 5-hydroxytryptamine  overnight at 4°C. Subsequently, the sections were incubated with goat anti-rabbit IgG HRP  or Alexa FluorH-test with post hoc Mann–Whitney U-test using IBM SPSS statistics software, ver. 25.0 . A value of p < 0.05 was considered to indicate statistical significance.The data are expressed as the mean ± standard deviation (SD). The statistical significance of differences between groups was evaluated by Kruskal–Wallis p < 0.01 for IS and p = 0.076 for UCMS in time in center, but not in locomotor activity), FST (p < 0.01 for both IS and UCMS in immobility and swimming time) and TST . Compared to UCMS, IS elicited notably worse depression-like behaviors, as evidenced by all three tests, the OFT, FST, and TST .J-SL wrote the main manuscript text, and conducted experiments. J-YK performed the statistical analysis and behavioral test, including tail suspension test, forced swimming test, and open field test. C-GS supervised the manuscript, and directed final version of all contents. All authors reviewed and approved this manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acquiring the optimum growth conditions of Ti-Al-N films, the effects of gas atmosphere, especially the reactive plasma on the material microstructures, and mechanical properties are still a fundamental and important issue. In this study, Ti-Al-N films are reactively deposited by radio frequency inductively coupled plasma ion source (RF-ICPIS) enhanced sputtering system. Different nitrogen gas flow rates in letting into the ion source are adopted to obtain nitrogen plasma densities and alter deposition atmosphere. It is found the nitrogen element contents in the films are quite influenced by the nitrogen plasma density, and the maximum value can reach as high as 67.8% at high gas flow circumstance. XRD spectra and FESEM images indicate that low plasma density is benefit for the film crystallization and dense microstructure. Moreover, the mechanical properties like hardness and tribological performance are mutually enhanced by adjusting the nitrogen atmosphere. Nowadlow rate . BecauseIn our previous work, radio frequency inductively coupled plasma ion source (RF-ICPIS) has been verified to enhance the Ti-Al-N deposition by dropping the argon gas ionization temperature and increasing the ionization rates . In this0.5Al0.5 compound target with purity of 99.9%. After pumping sputtering chamber to a base pressure lower than 1.0 × 10−4 Pa and target surface cleaning by argon ions, the buffer layer of Ti-Al was then deposited on substrates by DC sputtering. Following, the nitrogen plasma produced from RF-ICPIS was introduced nearby the substrates to attend the reactive deposition for Ti-Al-N film. The RF-ICPIS power was controlled at 50 W, and nitrogen gas flow rates in letting into the ion source were varied from 5 to 25 sccm to alter nitrogen plasma densities and obtain different sputtering and deposition atmosphere. Meanwhile, the DC sputtering current was fixed at 0.4 Å, and the total gas pressure of sputtering chamber was kept at 0.5 Pa. Detailed growth parameters are listed in Table Ti-Al-N films were deposited on Si 100) and mirror-polished stainless steel by RF-ICP ion source enhanced magnetron sputtering system with the growth temperature at 200 °C. Substrates were placed on a rotating specimen holder (20 rpm) which was perpendicular to a Ti00 and miλ = 1.54056 Å). Surface morphologies and cross-sectional microstructure of films were measured by field emission scanning electron microscopy . Atomic force microscopy  was applied to measure the surface root-mean-square (RMS). Pyramidal diamond tip was adopted for the nano-indentation test, and the hardness was measured by MTS Nano Indenter XP and calculated through the Oliver-Pharr indentation method. Each samples, ten separated point measurements, were taken to get a mean hardness value. A conventional ball-on-disc wear apparatus with a sliding speed of 0.2 m/s under a load of 10 N was used to measure the friction coefficient.Elements contents were characterized by energy dispersive spectrometer , and crystal structures of Ti-Al-N films were revealed by x-ray diffraction  with a Cu Kα radiation  wafers. As shown in Fig. 16 to 2.7 × 1016 cm−3 with the nitrogen gas flow rates increasing from 5 to 25 sccm. The improved electron concentrations indicate the plasma densities are improved during deposition progress. Additionally, the voltages of the sputtering source with a fixed sputtering current of 0.4 A during deposition are 482, 461, 443, 408, and 376 V for 5, 10, 15, 20, and 25 sccm respectively. The diminution of the impedance between substrates and target also reflect the enhanced plasma atmosphere. High plasma density improves the collisions between atoms that led the mean free path of sputtered metal atoms and film deposition rates decrease. Followed, the adsorbed atoms on the substrates have more time to migrate and nucleate and contribute to the film crystallizations. When nitrogen flow rates exceed a certain threshold, although the plasma densities are further improved, more nitrogen atoms would not be fully ionized under a fixed ion source power of 50 W [Degenerated Ti-Al-N crystal quality at high nitrogen gas flow rates is the result of nitrogen plasma variation originating from RF-ICPIS. More nitrogen atoms can be ionized when increase the nitrogen flow rates into the ion source, and the corresponding plasma densities in sputtering chamber are also enhanced. The electron concentrations near the substrates are measured by Langmuir probe. Calculated results show that the mean electron concentrations vary from 1.5 × 10Figure Further, the roughness of Ti-Al-N films deposited at various nitrogen gas flow rates is studied by AFM, and the root-mean-square (RMS) roughness values are plotted in Fig. The hardness of Ti-Al-N films deposited on stainless steel substrates under different nitrogen gas flow rates are shown in Fig. Friction properties of Ti-Al-N films are also studied by the ball-on-disc wear apparatus, and the mean friction coefficient of Ti-Al-N films is plotted in Fig. In summary, the microstructure and mechanical properties of Ti-Al-N films deposited at different nitrogen plasma atmosphere are studied. It is found that the element contents in the Ti-Al-N films are quite influenced by the nitrogen plasma density. Low plasma density is benefit for the film crystallization qualities and microstructure, which is supported by XRD spectra and FESEM. Additionally, the surface roughness and mechanical properties like hardness and friction coefficient can be further optimized at appropriate plasma density range. At high plasma density, the excessive nitrogen contents in the films can induce metastable nitride phase, and be responsible for the loose microstructure and aggravated mechanical performance. Our results would be an efficient way to further understand the deposition atmosphere-related growth mechanism of Ti-Al-N films.Additional file 1: Fig. S1. (a) Planar, (b)cross-sectional FESEM images and (c) friction coefficient curve of CS3, which are deposited through the traditional method of ionizing argon gas and has the same experimental parameter with S3.


Identification of SARS-CoV-2-infected patients at high-risk of poor prognosis is crucial. We aimed to establish predictive models for COVID-19 pneumonia severity in hospitalized patients.high-flow-oxygen-support were generated, one using baseline data and another with also follow-up analytical results. Calibration was performed by a 1000-bootstrap replication model.Retrospective study of 430 patients admitted in Vall d’Hebron Hospital (Barcelona) between 03-12-2020 and 04-28-2020 due to COVID-19 pneumonia. Two models to identify the patients who required high-flow-oxygen-support. The baseline predictive model showed a ROC of 0.800 based on: SpO2/FiO2 (adjusted Hazard Ratio-aHR = 8), chest x-ray (aHR = 4), prior immunosuppressive therapy (aHR = 4), obesity (aHR = 2), IL-6 (aHR = 2), platelets (aHR = 0.5). The cut-off of 11 presented a specificity of 94.8%. The second model included changes on the analytical parameters: ferritin (aHR = 7.5 if ≥200ng/mL) and IL-6 (aHR = 18 if ≥64pg/mL) plus chest x-ray (aHR = 2) showing a ROC of 0.877. The cut-off of 12 exhibited a negative predictive value of 92%.249 were male, mean age 57.9 years. Overall, 135 (31.4%) required high-flow-oxygen-support that may benefit from a more intensive disease management.SpO2/FiO2 and chest x-ray on admission or changes on inflammatory parameters as IL-6 and ferritin allow us early identification of COVID-19 patients at risk of  COVID-19, the disease caused by the novel virus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a global public health threat. Since December 2019 .Multiorgan damage might appear in severe COVID-19 cases. This syndrome is triggered by a dysfunctional host immune response to the virus that may persist in a context similar to the cytokine release syndrome –6. IncreThis study aimed to establish predictive models for disease severity based on baseline clinical and laboratory parameters, and thus, early identification of patients with SARS-CoV2 pneumonia who may benefit from a more intensive management, in order to ameliorate the course of the disease and reduce mortality.We included all consecutive adult patients hospitalized at the Internal Medicine wards due to COVID-19 pneumonia at Vall d’Hebron University hospital in Barcelona between 03/12/2020 and 04/28/2020. All patients had confirmed SARS-CoV-2 infection by RNA polymerase chain reaction (rtPCR) of nasopharyngeal swabs. In all cases demographic, clinical history and symptoms, radiological and laboratory data were collected from electronic medical records. All tests were performed according to the clinical care needs of the patient. Obesity was defined according to the WHO criteria as a BMI higher than 30. Radiologic assessment included always chest radiography and, in some cases, thoracic computed tomography (CT). Radiologic abnormality was determined by reviewing radiologic images by two attending physicians who extracted the data. In case of disagreement between the two reviewers a third physician reviewed the images. Radiologic findings were classified into: peripheral infiltrate, unilateral pneumonia and bilateral pneumonia. Unilateral pneumonia was defined if an infiltrate was observed in only one of the lungs. Peripheral infiltrate was described when involvement of the subpleural area was mainly present. Bilateral pneumonia was considered if diffuse infiltrates were present in both hemithorax.high-flow-oxygen support. This variable included all patients who required one or more of these conditions in order to maintain oxygen saturation over 90%: invasive mechanical ventilation, non-invasive mechanical ventilation, NHFC and non-rebreathing oxygen mask with reservoir. These mechanisms were used in a sequential way to obtain oxygen saturation over 90% in the case that the patient required a FiO2 higher than 50%.Due to the complex situation with limited resources during the pandemic peak, number of patients admitted to the intensive care unit or with access to non-invasive mechanical ventilation or nasal high-flow cannula (NHFC) was restricted. For this reason, in order to ascertain the prognosis of patients, and based on the view that some treatments such as corticosteroids are only beneficial for patients who need respiratory support , we geneLaboratory findings included complete blood count, electrolytes, liver and renal function tests and acute phase reactants including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), fibrinogen, D-dimer, lactate dehydrogenase (LDH), ferritin and IL-6. Blood tests were performed at admission, and at days 3 to 5, and 7 to 10 after admission in case of prolonged hospitalization. IL-6 was determined to indicate the use of some treatments, mainly Tocilizumab (TCZ).Treatment of severe COVID19 has changed in our center during the course of the pandemic and adapted to the most up-to-date clinical evidence. At the beginning of the pandemic the majority of patients received lopinavir/ritonavir (LPV/r) and hydroxychloroquine (HCQ) alone or in combination. In some patients with severe disease, immune modulatory drugs were used empirically, mainly corticosteroids and/or anti-IL-6R (TCZ). Pulses of methylprednisolone were defined as doses ≥1 mgr/kg. TCZ was considered if IL-6 was higher than 40pg/dL and SpO2/FiO2 (SaFi) below 250 or in the event of clinical worsening. A single intravenous dose of 400 or 600mg of TCZ depending on the weight of the patient (≥75Kg) was administered. This study was approved by the Ethics Committee of the Vall d’Hebron Hospital (PR(AG)242/2020). Written informed consent was waived.high-flow-oxygen-support, multivariate COX proportional hazard models was applied. We developed a model exclusively with baseline features. The second model included also follow up analytical data from day 3–5 after admission instead of baseline, for those patients who did not meet the outcome prior to that time. Variables showing a clinically and statistically significant association to the outcome in univariate analysis (Mantel-Cox- test) were selected for the initial models (p<0.10). The final models were fitted by using a step-wise forward method based on model Likelihood Ratios (Cox regression) with the same significance level (p<0.005) for entering and dropping variables. Quantitative variables included in the models were categorized  in order to increase the potency of the models. So as to estimate the performance of these models, calibration with a 1000-bootstrap samples was carried out to decrease the overfit bias [high-flow oxygen support was selected considering the highest Youden’s index, and shown by the sensitivity, specificity and predictive values. The results were considered statistically significant when the p-value was lower than 0.05. All statistical analyses were performed using IBM SPSS, version 26.0 .Normally distributed quantitative variables were expressed as mean and standard deviation (SD), and compared with Student t test. Quantitative variables non-normally distributed were expressed as median and interquartile range (IQR) and analysed with the Mann-Whitney U test. Categorical variables were expressed as frequency and percentage and compared using the chi-squared or Fisher’s exact test, when frequencies were less than 5%. In order to help early identification of patients at high-risk of poor respiratory prognosis, assessment by the combined variable fit bias , 14. FinVall d'Hebron ethics committee approved the study for PI20/00416 project.Oral consent was obtained from the patients in the context of the pandemic and recorded in their electronic clinical history. Patients' medical records were accessed between May to August of 2020 to obtain clinical data collection that was fully anonymized before analysed.Four hundred and thirty consecutive patients with pneumonia and COVID-19 infection confirmed by rtPCR were included. The majority of them were male (57.9%), mean age 57.9 years and 253 (58.8%) presented any comorbidities with arterial hypertension (39.3%) the most common. Obesity was present in 26.9% of the cohort, and underlying chronic obstructive pulmonary disease in 47 subjects (10.9%). At admission, 24 (5.6%) patients were on immunosuppressive therapy, mainly due to solid-organ transplant .The most frequent symptoms were fever in 342 (79.5%) and cough in 77.9% of patients. Median days from fever and cough onset to the admission were 6.3 and 7.1 days, respectively. Up to 57.9% of patients also complained of dyspnoea at admission. Other symptoms, like diarrhoea, myalgia and anosmia were less frequent . All patients presented an abnormal chest x-ray, with peripheral infiltrate as the most common finding (46.1%), followed by bilateral pneumonia (36%) and unilateral pneumonia (17.9%).At admission, 297 patients (69.1%) had an oxygen saturation over 95% and did not need oxygen support, whereas 54 patients (12.6%) required oxygen support over 0.31 litters per minute. Median ratio between oxygen saturation and oxygen requirement (SpO2/FiO2) was 399. A summary of patients’ characteristics at admission to hospital is shown in high-flow-oxygen-support, 28 (20.9%) within the first 24 hours of hospitalization. Median time from hospital admission to requirement of high-flow oxygen support was 3 days (IQR 1–5), with the majority of patients requiring 2 (N = 61) or 3 (N = 37) high-flow oxygen support devices sequentially . Yet, time from the beginning of dyspnoea was similar between the two cohorts (p = 0.200). Non-respiratory symptoms, like diarrhoea and myalgia were less frequently reported in those who later required high-flow oxygen support .high-flow-oxygen-support. Up to 79% of patients who did not require oxygen support at the emergency department would not need high-flow-oxygen-support during hospitalization. Importantly, radiologic findings were also predictive of poor prognosis, with up to 60% of patients with high-flow oxygen support presenting to hospital with bilateral pneumonia. Although patients with bilateral pneumonia were more prone to present at hospital with lower SpO2/FiO2, up to 53.5% of patients with baseline bilateral pneumonia did not need oxygen support at admission to hospital due to SpO2 >95%. Low SpO2/FiO2 ratio at admission was associated to early requirement of high-flow-oxygen-support presented with higher levels of ferritin , CRP , LDH , urea , and IL-6 .Regarding laboratory findings, patients who needed high-flow oxygen support had a lower lymphocyte, higher neutrophil count as well as a higher neutrophil/lymphocyte ratio . Individuals with Treatment characteristics are reflected in high-flow oxygen support or died during admission, because of the COVID-19 infection, respectively. There was no difference in the mortality rate according to the administration or not of TCZ .All patients included had at least one available value of serum IL-6. Two hundred and seventy-seven (64.4%) presented at any point IL-6 values > 40 pg/dL, though only 120 patients (27.9%) received treatment with TCZ since criteria for this treatment in our centre was also SaFi < 250. Among the 277 patients with at least one value of IL-6 > 40 pg/dL, 123 (44.4%) and 18 (6.5%) required Fifty three (12.3%) subjects received therapy with corticosteroid at doses higher than 1 mgr/kg for 3 or more days. The relatively low number of patients treated with corticosteroids can be explained by the fact that this drug was recommended by the COVID-19 Committee of our centre only in selected cases when this study was performed. Among these patients, 38 (71.7%) required high-flow oxygen support. In order to assess the possible impact of corticosteroids therapy, time from admission to hospital to the highest FiO2 needed by each patient was calculated.high-flow oxygen support was statistically lower in those who underwent “early corticosteroids”  when contrasted to those who received this therapy when they were already on their highest FiO2 requirement. In our opinion, this data supports the need of early administration of corticosteroids in order to modify the outcome of patients with COVID-19 infection.Moreover, a new variable named “early corticosteroids” was defined in those who began corticosteroids prior to the requirement of their highest FiO2. The vast majority of subjects were already on their highest FiO2 support when corticosteroids were initiated . Interestingly, the rate of patients who finally required Since only patients with severe COVID-19 have access to anti-inflammatory therapy, more patients in the group of high-flow oxygen support were treated with immunomodulatory therapy like anti-IL-6R therapy  or corticosteroids pulses , but this difference is due to a selection bias of the patients that were more severely ill. Although treatment had no effect on the need of high-flow oxygen support (p = 0.866), there was a tendency to lower mortality among those treated with the combination of LPV/r plus HCQ (p = 0.077) . Thus, ahigh-flow-oxygen-support 15.6% vs 1%, p<0.001). Univariate and multivariate analysis associated with mortality due to SARS-CoV-2 are shown in Median days of hospitalization were 12.2, with longer hospitalization in those with high-flow oxygen support . Overall mortality rate was 7.9% (34 deaths), being higher in patients with high-flow oxygen support . When only mortality directly related to COVID-19 was considered, the same difference was observed .As shown in COVID-19, a novel coronavirus disease spread massively throughout the world in few months, has been responsible for millions of cases and thousands of deaths worldwide. Roughly 6% of infected patients will progress to severe disease, and, in our study up to 30% of these required high-flow oxygen support, compromising the availability of health resources. In this setting, early identification of patients at risk of developing a severe state is essential in order to apply treatments that could potentially impact on the disease progression.To date, a few indexes are used , few of One of the distinctive aspects and strengths of our study is the categorization of comorbidities. Previous models give the same weight to all comorbidities , 17, losIn our cohort, although some comorbidities such as obesity, diabetes, hypertension, COPD and immunosuppression were associated with a higher rate of high-flow oxygen support, only obesity and immunosuppressive therapy were independently linked with the composite endpoint. Interestingly, clinical presentation differed in patients that later required or not of high-flow oxygen support. Dyspnoea was more commonly reported by those who needed this oxygen support, but non-respiratory findings such as myalgias and diarrhoea were more frequent among patients with good prognosis. In fact, the possible positive impact of diarrhoea in patients with COVID-19 should be highlighted, since a tendency towards higher frequency of this symptom was observed among patients without high-flow oxygen requirement. It may be worth exploring this negative association on an independent cohort with a larger number of patients.Despite the fact many laboratory parameters have been previously related to poor outcomes, such as the neutrophils/lymphocyte ratio, urea, LDH, platelets and acute phase reactants such as ferritin, procalcitonin and CRP , the anaInterestingly, in the follow up model the only factors independently associated with the need of high-low oxygen support were chest x-ray and increase in inflammatory parameters such as IL-6 and ferritin from day 0 and days 3–5, yielding to a model with a high performance (R = 0.877). This emphasizes the importance of inflammatory determinants at baseline and throughout the course of the disease. As aforementioned, the chosen outcome of our predictive models was the requirement of high-flow oxygen support.Although previous studies have mainly focused on COVID-19 mortality and/or intensive care unit (ICU) admission , 19, theOur study presents some limitations. This is a retrospective and unicentric cohort. Though characterization of comorbidities was thorough, predictive factors in the predictive models varied due to the different number of patients in both models, due to loss of patients  and also, but to a lesser extent, due to lack of follow up IL-6 or ferritin values in some patients.Nevertheless, the models were calibrated by 1000-bootstrap samples, and the main factors associated with the need of high-flow oxygen support were consistent in both models (baseline chest x-ray and IL-6). Another strength of our study is the still low number of reports on COVID-19 predictive prognostic scores in non-Asian population.Moreover, in our study the outcome of patients has been assessed in terms of respiratory support, including not only invasive mechanical ventilation, but all types of high-flow devices, leading to a more realistic overview of the COVID-19 severity than those scores just focusing on the need for ICU admission or mortality. Since the SARS-CoV-2 had just been identified at the time of our study, kinetics of viral load and antibody titres anti-SARS-CoV-2 were not available.In conclusion, a third of patients admitted to the hospital with COVID-19 needed high-flow oxygen support which is associated with a worse prognosis. This study provides predictive models for disease severity based on baseline characteristics, allowing an early identification of those patients who can benefit from a more intensive disease management in order to prevent or reduce COVID-19 complications.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file. 8 Nov 2020PONE-D-20-30311Simple predictive models identify patients with COVID-19 pneumonia and poor prognosisPLOS ONEDear Dr. Labrador-Horrillo,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. 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Reviewer #1: This study aimed to establish predictive models for COVID-19 pneumonia severity in hospitalized patients.The authors attempted to build predictive models to identify the patients who required high-flow-oxygen-support .My major concerns are that low SpO2/FiO2 and abnormal chest x-ray are significant factors in the models.In the causal model, significant pneumonia may lead to low SpO2/FiO2, which may increase the use of high flow oxygen support. SpO2/FiO2 would be an intermediate variable which should not be added in the models for adjustment. I suggest the authors build the predictive models without SpO2/FiO2 to evaluate if the AUC is still acceptatble.Reviewer #2: The article ‘Simple predictive models identify patients with COVID-19 pneumonia and poor prognosis' presents a predictive model for COVID-19 pneumonia in hospitalized patients. The predictive model showed a ROC of 0.800 based on: SpO2/FiO2 (adjusted Hazard Ratio-aHR:8), chest x-ray (aHR:4), prior immunosuppressive therapy (aHR:4), obesity (aHR:2), IL-6 (aHR:2), platelets (aHR:0.5) by the analysis of 430 patients admitted in Vall d’Hebron Hospital (Barcelona). This is a very important data in the pandemic of COVID-19. My comments are below:Major Comments1. Criteria of induction of high-flow oxygen supportResults included some analysis of the patient's background using high-flow oxygen support. For example, they were bilateral abnormal shadows in a chest X-ray image and a decrease in the number of lymphocytes. On the other hand, since the results are from a single center, there may be a protocol for induction of high-flow oxygen support. First, please describe the criteria for introduction of high-flow oxygen support. The information will be important information for evaluating the predictive models.２．Predictive score of induction of high-flow oxygen support in COVID-19　Age, medical past history , NLR, CRP, ferritin and LDH were with significant difference in induction of high-flow oxygen support. Is it possible to statistically calculate the predictive score of whether to introduce high-flow oxygen support in COVID-19? We would like to ask if it is possible to predict X% for 3 out of 6, Y% for 4, Z% for 5, and so on.Reviewer #3: Authors conducted a retrospective study of 430 patients admitted in Vall d’Hebron Hospital (Barcelona) between 03-12-2020 and 04-28-2020 due to COVID-19 pneumonia and would like to create a model to identify risk factors in patients with high-flow-oxygen-support group, they identify SpO2/FiO2 and chest x-ray on admission or changes on inflammatory parameters as IL-6 and ferritin allow us early identification of COVID-19 patients at risk of high-flow-oxygen-support that may benefit from a more intensive disease management.Some concerns need to clarify.Major1. Authors said 135 patients (31.4%) needed high-flow-oxygen-support after admission. How many patients who initially had SpO2 >95% but finally needed High-flow-oxygen-support, any risk factors about progress to desaturation in the subgroup? Associated with treatment options?2. How to distinguish “peripheral infiltrate” from “unilateral pneumonia “or “bilateral pneumonia”? in clinical, peripheral infiltrate belongs to the above. Do authors define pneumonia is consolidation or air space shadow instead of infiltrates?3. Figure 1A. Why up to 28.9% patients with high flow oxygen support didn’t receive any therapy such as LPV/r plus HCQ or HCQ in your cohort, the treatment options may seriously affect outcome and made the risk factors of poor prognosis became not reliable. Authors should compare the efficacy in different treatment subgroups in patients with high-flow-oxygen-support.4. How to decide these patients with high-flow-oxygen-support had to receive LPV/r plus HCQ or HCQ or none? In figure 1B, authors showed the time of hospitalization in different treatment arms, however, the time may be influenced by death. Therefore, authors are encouraged to define other endpoints, such as the time to leave High-flow-oxygen-support or successful discharge.5. Please define the “obesity” since obesity is a significant risk factor in multivariable COX regression model in the study.6. The role of serum IL-6 in your cohort? A prognostic factor or indicator for drug selection? Any different outcome in patients with IL-6 higher than 40pg/dL and SpO2/FiO2 (SaFi) below 250 received or not received TCZ in your cohort? All high serum IL-6 patients received TCZ, or combination therapy?7. How many patients received corticosteroid in your cohort since several studies in JAMA and NEJM indicated corticosteroid administration may have better outcome.8. No patients received redemsivir, the only FDA approval emergent use drug for COVID-19, in your cohort?MinorPage 6, line 21, hidroxychloroquine --> hydroxychloroquinePage 8, line 14, chronic pulmonary disease --> chronic obstructive pulmonary disease**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  17 Nov 2020#Journal requirements:1- Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfWe have ensured that our manuscript meets PLOS ONE's style requirements2. In the ethics statement in the manuscript and in the online submission form, please provide additional information about the patient records used in your retrospective study, including: a) whether all data were fully anonymized before you accessed them; b) the date range (month and year) during which patients' medical records were accessed. If patients provided consent to have data from their medical records used in research, please include this information, including: whether consent was informed and what type you obtained .We have corrected the ethics statement accordingly and added it in the manuscript.3. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 5 in your text; if accepted, production will need this reference to link the reader to the Table.Table 5 is correctly cited in the “Results” section located now in Page 13 line 17-18. We have highlighted it in yellow in the manuscript.http://journals.plos.org/plosone/s/supporting-information.4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: We added “Supporting Information” files at the end of the manuscript.#Reviewers' comments:Reviewer #1:This study aimed to establish predictive models for COVID-19 pneumonia severity in hospitalized patients. The authors attempted to build predictive models to identify the patients who required high-flow-oxygen-support. My major concerns are that low SpO2/FiO2 and abnormal chest x-ray  are significant factors in the models. In the causal model, significant pneumonia may lead to low SpO2/FiO2, which may increase the use of high flow oxygen support. SpO2/FiO2 would be an intermediate variable which should not be added in the models for adjustment. I suggest the authors build the predictive models without SpO2/FiO2 to evaluate if the AUC is still acceptable.We appreciate the reviewer’s suggestion. Although patients with bilateral pneumonia were more prone to present at hospital with lower SpO2/FiO2, up to 53.5% of patients with baseline bilateral pneumonia did not need oxygen support at admission to hospital due to SpO2 >95%. A new S3 Table summarized the need of oxygen supplementation at admission to the ER department and the mean SpO2/FiO2 according to the baseline chest X-ray findings.This data has been added to the revised version of the manuscript in order to improve the quality of the paper (Page 10 lines 11 – 15).Regarding the causal model, though as previously mentioned chest X-ray was associated with lower SpO2/FiO2, both were independent factors associated with the need of high-flow oxygen support, and for this reason they were included in the predictive model. However, as suggested by the reviewer, we conducted a new multivariate analysis excluding the variable “SpO2/FiO2”, turning out the following predictive model:This model yields a ROC of 0.710 (0.650-0.770), value lower than the ROC showed by the predictive model including SpO2/FiO2 (ROC 0.800).This new analysis may be added to the manuscript as supporting information data if the reviewer or editor thinks it would be suitable for readers. Reviewer #2:The article ‘Simple predictive models identify patients with COVID-19 pneumonia and poor prognosis' presents a predictive model for COVID-19 pneumonia in hospitalized patients. The predictive model showed a ROC of 0.800 based on: SpO2/FiO2 (adjusted Hazard Ratio-aHR:8), chest x-ray (aHR:4), prior immunosuppressive therapy (aHR:4), obesity (aHR:2), IL-6 (aHR:2), platelets (aHR:0.5) by the analysis of 430 patients admitted in Vall d’Hebron Hospital (Barcelona). This is a very important data in the pandemic of COVID-19. My comments are below:1. Criteria of induction of high-flow oxygen supportResults included some analysis of the patient's background using high-flow oxygen support. For example, they were bilateral abnormal shadows in a chest X-ray image and a decrease in the number of lymphocytes. On the other hand, since the results are from a single center, there may be a protocol for induction of high-flow oxygen support. First, please describe the criteria for introduction of high-flow oxygen support. The information will be important information for evaluating the predictive models.Thank you very much for your comment. Related to the oxygen support, the target was to obtain an oxygen saturation higher than 90-92%, or a SaFi higher than 250. In this sense, when the patient required a FiO2 higher than 50%, a non-rebreathing oxygen mask with reservoir, nasal high-flow cannula, non-invasive mechanical ventilation, and invasive mechanical ventilation were used sequentially. To clarify this point a sentence has been added to the manuscript: Page 6 lines 14 – 15: “These mechanisms were used in a sequential way to obtain oxygen saturation over 90% in the case that the patient required a FiO2 higher than 50%”.2. Predictive score of induction of high-flow oxygen support in COVID-19, age, medical past history , NLR, CRP, ferritin and LDH were with significant difference in induction of high-flow oxygen support. Is it possible to statistically calculate the predictive score of whether to introduce high-flow oxygen support in COVID-19? We would like to ask if it is possible to predict X% for 3 out of 6, Y% for 4, Z% for 5, and so on. We appreciate the reviewer’s comment. Since all the factors included in the multivariate analysis are quite common and frequently available, we constructed the baseline predictive model with adjusted coefficients after a 1000-bootstrap samples calibration, the latter as a strategy to avoid the bias of the lack of a validation cohort.Absence of data of any of the factors, i.e. analytical parameters such as IL-6 and platelets, would indeed decrease the performance of the model, though it still achieves a moderate ROC, with ROC of 0.789 (95% CI 0.740-0.839) and 0.774 (95% CI 0.723-0.825), for the model without IL-6 and without both IL-6 and platelets values, respectively.Nevertheless, since we realized that exclusion of one or more factors may change the predictive model, we calculated the new coefficients after discarding the IL-6 plus/minus platelets values , resulting in the following coefficients:- Baseline model without IL-6: Immunosuppression * 4.372 (Hazard Ratio-HR) + Chest x-ray (3 categories) * 1.924 + SpO2/FiO2 (4 categories) * 4.593 + Obesity * 1.801 + Platelets (>215x10E9/mL) * 0.558.- Baseline model neither IL-6 nor platelets: Immunosuppression * 3.816 + Chest x-ray (3 categories) * 1.929 + SpO2/FiO2 (4 categories) * 4.371 + Obesity * 1.714.The ROC of the aforementioned new baseline models are shown in figures included in the letter in response to reviewers.These new analyses may be added to the manuscript as supporting information data if the reviewer or editor thinks it would be suitable for readers. Reviewer #3:Authors conducted a retrospective study of 430 patients admitted in Vall d’Hebron Hospital (Barcelona) between 03-12-2020 and 04-28-2020 due to COVID-19 pneumonia and would like to create a model to identify risk factors in patients with high-flow-oxygen-support group, they identify SpO2/FiO2 and chest x-ray on admission or changes on inflammatory parameters as IL-6 and ferritin allow us early identification of COVID-19 patients at risk of high-flow-oxygen-support that may benefit from a more intensive disease management. Some concerns need to clarify.1. Authors said 135 patients (31.4%) needed high-flow-oxygen-support after admission. How many patients who initially had SpO2 >95% but finally needed High-flow-oxygen-support, any risk factors about progress to desaturation in the subgroup? Associated with treatment options? We agree with the reviewer that analysis of patients with baseline SpO2 >95% is very interesting. As shown in Table 1, at admission to hospital 297 (69.1%) of the patients presented SpO2 >95% and therefore they didn’t need oxygen support at that time. However, 63 (21.2%) required high-flow-oxygen-support later on during admission to hospital. In line with overall results from our cohort, and more than probably due to a bias selection and late administration, patients treated with either TCZ or corticosteroids presented a statistically higher need of high-flow-oxygen-support . Nevertheless, lack of therapy in comparison with HCQ alone or in combination with LPV/r had no impact on prognosis in terms of high-flow-oxygen-support . In this subgroup of patients, the univariate analysis showed an association between the following factors and later need of high-flow-oxygen-support: diabetes, obesity, days of fever, days of dyspnea, chest CX-ray, baseline lymphocyte and platelets account, fibrinogen and urea. However, the multivariate analysis revealed that baseline chest x-ray was the only predictor independently associated with later poor prognosis . This data has been added to the revised version of the manuscript in order to improve the quality of the paper. “Page 13 line 19 – Page 14 line 2”2. How to distinguish “peripheral infiltrate” from “unilateral pneumonia “or “bilateral pneumonia”? in clinical, peripheral infiltrate belongs to the above. Do authors define pneumonia is consolidation or air space shadow instead of infiltrates?We highly appreciate the comments of the reviewer. As we state in the manuscript, chest radiography was interpreted by two attending physicians. Unilateral pneumonia was defined if an infiltrate was observed in only one of the lungs. Peripheral infiltrate was described when involvement was mainly present in the subpleural area. Bilateral pneumonia was considered if diffuse infiltrates were present in both hemithorax. This explanation has been added to the main text in order to clarify concepts. Page 5 line 25 – Page 6 line 43. Figure 1A. Why up to 28.9% patients with high flow oxygen support didn’t receive any therapy such as LPV/r plus HCQ or HCQ in your cohort, the treatment options may seriously affect outcome and made the risk factors of poor prognosis became not reliable. Authors should compare the efficacy in different treatment subgroups in patients with high-flow-oxygen-support.We appreciate the kind comments of the reviewer. At the time that patients were collected, between the beginning of March and the end of April 2020, the standard of care in our hospital for patients affected by COVID19 was HCQ plus LPV/r, both alone or in combination. However, pharmacological interactions were very frequent with these drugs and other agents. On the other hand, side effects, like diarrhea and QT enlargement, mostly related to LPV/r were present in many patients, and for this reason these drugs were stopped early on. Fortunately, different studies have discarded any beneficial role of these treatments in patients with COVID19, and for this reason they are no longer used.4. How to decide these patients with high-flow-oxygen-support had to receive LPV/r plus HCQ or HCQ or none? In figure 1B, authors showed the time of hospitalization in different treatment arms, however, the time may be influenced by death. Therefore, authors are encouraged to define other endpoints, such as the time to leave High-flow-oxygen-support or successful discharge. We appreciated the reviewer’s observation. As summarized in the Materials and methods section and commented previously in this response, according to our centre protocol for treatment of COVID19, all admitted patients with pneumonia received treatment with LPV/r plus/minus HCQ, except contraindication, drug-drug interaction or intolerance. We agree with the reviewer that it would be interesting to assess the possible impact of pharmacological treatment on time to successful discharge. There was no difference in the mean time of admission according to the received treatment These last data have been added to figure 1B in order to highlight successful discharge.5. Please define the “obesity” since obesity is a significant risk factor in multivariable COX regression model in the study.According to WHO, obesity was defined as a BMI higher than 30. This has been added to in the Materials and methods section. “Page 5 lines 19 – 20”6. The role of serum IL-6 in your cohort? A prognostic factor or indicator for drug selection? Any different outcome in patients with IL-6 higher than 40pg/dL and SpO2/FiO2 (SaFi) below 250 received or not received TCZ in your cohort? All high serum IL-6 patients received TCZ, or combination therapy?All patients included had at least one available value of serum IL-6. Two hundred and seventy-seven (64.4%) presented at any point IL-6 values > 40 pg/dL, though only 120 (27.9%) received treatment with TCZ since criteria for this treatment in our centre was also SaFi < 250. Among the 277 patients with at least one value of IL-6 > 40 pg/dL, 123 (44.4%) and 18 (6.5%) required high-flow oxygen support or died during admission, because of the COVID-19 infection, respectively. There was no difference in the mortality rate according to the administration or not of TCZ . However, need of high-flow oxygen support was statistically higher among those treated with TCZ in contrast with those who did not undergo this therapy . As discussed in the manuscript: Page 12 lines 3 – 7 “Since only patients with severe COVID-19 have access to anti-inflammatory therapy, more patients in the group of high-flow oxygen support were treated with immunomodulatory therapy like anti-IL-6R therapy  or corticosteroids pulses , but this difference is due to a selection bias of the patients that were more severely ill.” Once again, this fact emphasized the need of early identification and anti-inflammatory treatment of patients at risk of poor prognosis.We added a paragraph to the manuscript to clarify the question: Page 11 lines 4 – 11.7. How many patients received corticosteroid in your cohort since several studies in JAMA and NEJM indicated corticosteroid administration may have better outcome.We appreciate the reviewer’s comment. Fifty three (12.3%) subjects received therapy with corticosteroid at doses higher than 1 mgr/kg for 3 or more days. The relatively low number of patients treated with corticosteroids can be explained by the fact that this drug was recommended only in selected cases by the COVID-19 Committee of our centre when this study was performed.Among these patients, 38 (71.7%) required high-flow oxygen support. In order to assess the possible impact of corticosteroids therapy, time from admission to hospital to the highest FiO2 needed by each patient was calculated. Moreover, a new variable named “early corticosteroids” was defined in those who began corticosteroids prior to the requirement of their highest FiO2. The vast majority of subjects were already on their highest FiO2 support when corticosteroids were initiated . Interestingly, the rate of patients who finally required high-flow oxygen support was statistically lower in those who underwent “early corticosteroids”  when contrasted to those who received this therapy when they were already on their highest FiO2 requirement. In our opinion, this data supports the need of early onset of corticosteroid in order to modify outcome of patients with COVID-19 infection. We added these new data to the manuscript Page 11 line 12 – Page 12 line 28. No patients received redemsivir, the only FDA approval emergent use drug for COVID-19, in your cohort?In our cohort just one patient received Remdesivir because its use was restricted in our hospital at that time, to ICU patients. This patient was admitted to ICU the same day he consulted to the ER department due to severe pneumonia that required HFNC during the first hours of admission.Page 6, line 21, hidroxychloroquine --> hydroxychloroquinePage 8, line 14, chronic pulmonary disease --> chronic obstructive pulmonary diseaseWe appreciate the reviewer’s comment. We have corrected these spelling errorsAttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 15 Dec 2020Simple predictive models identify patients with COVID-19 pneumonia and poor prognosisPONE-D-20-30311R1Dear Dr. Labrador-Horrillo,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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